Cellular immune responses are often initiated by T cells bearing αβ-T cell receptors (TCRs) which typically recognize foreign peptides bound to classical major histocompatibility complex (MHC) molecules on specialized antigen presenting cells (Zinkernagel and Doherty, 1997). There are two classes of MHC molecules—MHC class I (MHC-I) and MHC class II (MHC-II). Within MHC-I, there are two subclasses, MHC-Ia (‘classical’ MHC) and MHC-Ib (‘non-classical’ MHC).
Major histocompatibility complex-related protein 1 (MR1) is a MHC class Ib molecule encoded by a single functional, monomorphic Mr1 gene in antigen presenting cells. The MR1 protein, like MHC class I, is comprised of a heavy chain (comprised of the α1, α2 and α3 domains) non-covalently associated with a light chain (β2-microglobulin). The Mr1 gene is not Mhc linked, is highly conserved, and seems to be unique to mammals. As striking evidence for interspecies conservation, the predicted amino acid sequences of mouse MR1 (mMR1) and human MR1 are 89/90% identical in their 1/2 domains. By contrast, mouse and human MHC-linked class Ia and Ib molecules are 69/70% and 51/41% identical, respectively. The high level of polymorphism of classical MHC molecules allows them to present diverse peptides to T cells during the adaptive immune response to pathogens. By contrast, the remarkable conservation of MR1 suggests that it evolved under strong negative selection, possibly imposed by immune responses to pathogens. MR1 message and protein are ubiquitously expressed in different tissues. Endogenous MR1 is only detected on the plasma membrane of cells from murine or human origins at very low levels using available monoclonal antibodies (mAbs) considered specific for MR1. However, higher levels of surface expression of MR1 can be achieved using transfection or transduction to overexpress an MR1-encoding cDNA in mouse or human cell lines. The failure to detect even moderate levels of endogenous MR1 at the cell surface is suggested to reflect limited ligand supply as is the case with the non-classical MHC, H2-M3, which presents N-formylated peptides.
MR1 cell surface expression is required for the in vivo development of a recently identified population of mucosal-associated T (MAIT) cells that are typically classified as possessing an invariant TCR-chain (i.e. identical V-J combination).
The importance of the role of MAIT cells in immunity is indicated by their conservation across species such as humans, cattle and mice, as well as recent data implying protective function in certain infections, e.g. in vivo pulmonary bacterial infections (Gold et al., 2010a; Le Bourhis et al., 2011; Le Bourhis et al., 2010, Mejerovics A et al., 2013; Serriari N E et al., (2014)) and inflammatory conditions including multiple sclerosis. In humans, MAIT cells comprise 1-10% of peripheral blood T cells when compared to their NKT cell counterparts (typically less than 0.1%) (Godfrey et al., 2010b). Indeed, MAIT cells are found in human blood, the gastrointestinal mucosa and mesenteric lymph nodes. Furthermore, MAIT cells, like NKT cells, rapidly produce a broad range of cytokines upon activation (Kawachi et al., 2006; Martin et al., 2009). There are further parallels between MR1-restricted MAIT cells and CD1d-restricted NKT cells in that, like NKT cells, MAIT cells typically express a semi-invariant TCR, comprised of an invariant TCR alpha-chain (V 19J 33 in mice or the homologous V 7.2J 33 in humans) in combination with TCR-V 6 or V 8 in mice and TCR-V 2 or V 13 in humans. Other alpha chains have been described, for example, Vα7.2 joined to Jα20 or Jα12 (Rentragoon et al (2013). The semi-invariant and evolutionarily conserved nature of the MAIT TCR suggests that MAIT cells are specific for an important, albeit limited and atypical, class of antigens (Ags) presented by the MR1 molecule. Further, evidence for a highly conserved MAIT-ligand comes from mutagenesis studies of MAIT TCRs with different Vβ-segments which have revealed that a defined cluster of amino acid residues are crucial for MAIT cell recognition of diverse microbes (Reantragoon et al, 2012). MAIT cells respond to a surprisingly broad range of microorganisms, excluding viruses but including diverse strains of bacteria and yeast, suggesting the existence of a conserved Ag (or family of Ag), common to these cellular organisms, presented to MAIT cells in an MR1-dependent manner (Gold et al., 2010a; Gold et al., 2010b; Le Bourhis et al., 2010). This suggests a much broader role in the immune response than is indicated by their limited TCR repertoire.
In humans, MAIT cells are traditionally defined as CD161hi, IL-18R, +V 7.2+, −CD3+ lymphocytes. Current methods of staining of MAIT cells in both peripheral blood and tissues require either staining for CD161 or IL-18R expression at the cell surface, together with staining of the V 7.2 segment (Martin et al, 2009; Le Bourhis et al, 2010). A key limitation of this phenotypic characterization of MAIT cells is that these cells may include T cells other than those expressing the V 7.2. Moreover, T cells that do express the V 7.2 also occur in the normal course of other immune responses including MHC-restricted responses and potentially other MHC1b-restricted immunity and therefore these V 7.2+ cells are unrelated to MAIT cell specificity. Hence, the monitoring and identification of MAIT cells by current techniques reliant entirely on a V 7.2 phenotype is subject to a significant ‘false-positive’ effect. In addition, the precise identification of MAIT cells in mice has been even more difficult, as they are traditionally defined using V 19 and V 6 or V 8.
Because of the emerging understanding of the role that MAIT cells play in the immune response, there is a need to identify the exact mechanisms by which MAIT cells exert their effects. This has been significantly hindered because prior to the present invention the precise identity of the MR1-restricted Ag(s) which represents a key step in understanding MAIT cell biology has been unknown.
Thus, there remains a need for new tools to specifically recognize MAIT cells in mammals, which would be useful, inter alia, for recognizing, purifying, and enriching these cells in vivo or in vitro, to allow the facilitation of methods of labeling MAIT cells for research and diagnostic purposes. There also remains a need to identify the ligand(s) bound by MR1, including determining the TCR antigen specificity of MAIT cells which will allow the facilitation of methods of modulating MAIT cell activity for therapeutic purposes.